Method for detecting target molecule

ABSTRACT

This invention provides a technique enabling to detect target molecules of low concentration with high sensitivity. This invention includes (i) a step of introducing a hydrophilic solvent ( 42 ) containing beads ( 40 ),( 41 ′) into a space ( 30 ) between (a) a lower layer section ( 10 ) including a plurality of receptacles ( 13 ) each of which is capable of storing only one of the beads ( 41 ),( 41 ′) and which are separated from each other by a side wall ( 12 ) having a hydrophobic upper surface and (b) an upper layer section ( 20 ) facing a surface of the lower layer section ( 10 ) on which surface the plurality of receptacles ( 13 ) are provided; and (ii) a step of introducing a hydrophobic solvent ( 43 ) into the space ( 30 ), the step (ii) being carried out after the step (i).

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of co-pending U.S. applicationSer. No. 15/351,522 having a filing date of Nov. 15, 2016, which is adivisional of then-co-pending U.S. application Ser. No. 15/082,195having a filing date of Mar. 28, 2016, now abandoned, which is adivisional of then-co-pending U.S. application Ser. No. 14/003,509having a § 371(c)(1), (2), (4) date of Sep. 6, 2013, now U.S. Pat. No.9,329,174, which is a U.S. national stage entry under 35 U.S.C. § 371 ofInternational Patent Application No. PCT/JP2012/055884 filed on Mar. 7,2012, which claims the benefit of foreign priority to Japanese PatentApplication No. JP 2011-050629 filed on Mar. 8, 2011, the disclosures ofall of which are hereby incorporated by reference in their entireties.The U.S. application Ser. No. 15/351,522 was published on Apr. 27, 2017,as US 2017/0115284 A1. The U.S. application Ser. No. 15/082,195 waspublished on Aug. 4, 2016, as US 2016/0223531 A1. The InternationalApplication was published in Japanese on Sep. 13, 2012, as InternationalPublication No. WO 2012/121310 A1 under PCT Article 21(2).

TECHNICAL FIELD

The present invention relates to a bead sealing method (i.e., a methodfor sealing beads), a method for detecting a target molecule, an array,a kit, and a target molecule detection device.

BACKGROUND ART

There has been known a single-molecule assay as a method for carryingout various assays by observing biomolecules such as proteins andnucleic acids in such a manner that the biomolecules are individuallyidentified. In order to carry out the single-molecule assay, there havebeen known some methods.

Patent Literature 1 discloses a micro chamber for detectingsingle-molecule enzyme activity. This micro chamber includes a containerpart into which a liquid droplet can be sealed and which has capacity ofstoring a liquid droplet of up to 1000 fL (femtoliters). The containerpart is made of a recess provided in at least one of a first member anda second member which are bonded to each other. According to PatentLiterature 1, an enzyme reaction is carried out in the liquid droplet.With such a configuration, the enzyme reaction can be performed with ahigh concentration of the reaction products, even if the number ofmolecules of the reaction products is quite small. Thus, it is possibleto detect an activity of one molecule of enzyme.

Non-Patent Literature 1 discloses a method for carrying out asingle-molecule enzyme assay by use of an array where a liquid dropletis covered with oil, in a femtoliter-order, and accessible directly fromthe outside. This array includes a hydrophilic region pattern made of ahydrophilic surface on which a hydrophobic region having a height of 17nm is provided.

Non-Patent Literature 2 discloses a method for detecting a protein by asingle-molecule Enzyme-Linked ImmunoSorbent Assay (ELISA). According tothis method, a very small amount of proteins are captured by minutebeads covered with protein-specific antibodies, and complexes of thebeads and the proteins are fluorescence-labeled. Then, beads includingthe complexes are introduced into a reaction chamber by centrifugalforce. Thereafter, the number of beads having captured the proteins iscounted. In this manner, the proteins are quantitatively assayed.

CITATION LIST Patent Literatures

[Patent Literature 1]

-   Japanese Patent Application Publication, Tokukai, No. 2004-309405 A    (Publication Date: Nov. 4, 2004)

Non-Patent Literatures

[Non-Patent Literature 1]

-   S. Sakakihara et al., Lab Chip, 2010, 10, 3355-3362

[Non-Patent Literature 2]

-   David M Rissin et al., Nature Biotechnology: doi: 10.1038/nbt. 1641

SUMMARY OF INVENTION Technical Problem

In order to detect, e.g., disease markers of low concentration for earlydetection of diseases, infectious diseases, and the like, there is ademand for biosensing techniques developed to have higher sensitivities.For example, in a case where one million cancer cells included in atumor having a volume of 1 mm³ secrete marker proteins (100 moleculesper cell) into 5-liter blood, a concentration of the proteins in theblood is approximately 30 aM (i.e., 30×10⁻¹⁸ M). A technique capable ofdetecting target molecules of such quite low concentration is needed.

A possible method for detecting such the target molecules may be the onefor detecting the target molecules by the above-mentionedsingle-molecule enzyme assay at a single molecule level sensitivity.Specifically, such the method is carried out by (i) sealing the targetmolecule specifically into a femtoliter-order liquid droplet (very smallliquid droplet), (ii) linking the target molecule to a substance such asan enzyme-labeled antibody, and (iii) detecting an activity of theenzyme labeling the antibody in the above-mentioned manner. The sealingof the target molecule specifically into the very small liquid dropletmay be carried out by a method using, e.g., a bead labeled with asubstance such as another antibody for specifically binding to thetarget molecule. In this method, after the bead is bound to the targetmolecule, the bead is sealed into the very small solution droplet.

Incidentally, in order to efficiently detect target molecules which arecontained in a solution only in a very small amount e.g., approximately30 aM target molecules as described above, it is necessary to prepare alarge number of very small liquid droplet arrays, as many asapproximately one million, and to cause the arrays to capture the beads.

However, according to the method disclosed by Non-Patent Literature 2,the beads need to be introduced into arrays by strong centrifugal force,and therefore much time and efforts are required. Further, the number ofarrays used in the method of Non-Patent Literature 2 is approximatelyfifty thousand. Therefore, the method of Non-Patent Literature 2 isquite difficult to be applied to the case requiring approximately onemillion arrays. Thus, with the method of Non-Patent Literature 2, it isdifficult to efficiently seal a large number of beads into the arrays.Incidentally, none of Patent Literature 1 and Non-Patent Literature 1discloses any method for solving such the problem.

In view of this, the present invention has an object to provide atechnique for efficiently sealing a large number of beads into an array.

Solution to Problem

In order to attain the above object, a method of the present inventionfor sealing beads includes: (i) a step of introducing a hydrophilicsolvent containing beads into a space between (a) a lower layer sectionincluding a plurality of receptacles each of which is capable of storingonly one of the beads and which are separated from each other by a sidewall having a hydrophobic upper surface and (b) an upper layer sectionfacing a surface of the lower layer section on which surface theplurality of receptacles are provided; and (ii) a step of introducing ahydrophobic solvent into the space, the step (ii) being carried outafter the step (i).

In order to attain the above object, an array of the present inventionincludes: a lower layer section provided with a plurality of receptaclesbeing separated from each other by a side wall having a hydrophobicupper surface; and an upper layer section facing, via a space, a surfaceof the lower layer section on which surface the plurality of receptaclesare provided.

Advantageous Effects of Invention

The use of the method for sealing beads of the present invention makesit possible to efficiently seal a large number of beads into an array,thereby contributing to a technique by which target molecules of lowconcentration are detectable with high sensitivity.

BRIEF DESCRIPTION OF DRAWINGS

(a) through (e) of FIG. 1 are views schematically illustrating a methodfor sealing beads according to the present invention, and show lateralcross-sectional views of an array 1.

FIG. 2 is a view schematically illustrating one embodiment of a targetmolecule detection device according to the present invention.

FIG. 3 shows a fluorescence image of an array into which beads weresealed in one example of the present invention.

FIG. 4 is a graph showing fluorescence intensities observed when targetmolecules were detected by a conventional method.

(a) through (f) of FIG. 5 show microscopic images of arrays into whichbeads were sealed in another example of the present invention.

FIG. 6 shows a graph illustrating a relationship, observed in saidanother example of the present invention, between (i) a concentration ofstreptavidin and (ii) a ratio of the number of beads having capturedstreptavidin with respect to the number of beads stored in the array.

FIG. 7 is a view for explaining a method for preparing ahydrophilic-hydrophobic patterned glass according to an example of thepresent invention.

FIG. 8 is a view illustrating (i) a bead trapping efficiency found in acase involving the use of an array having a flow cell structure (Example3) and (ii) a bead trapping efficiency found in a case involving the useof an array not having the flow cell structure (Comparative Example 2).

DESCRIPTION OF EMBODIMENTS

The following describes one embodiment of the present invention indetails.

[Method for Sealing Beads]

With reference to (a) through (e) of FIG. 1, the following describes amethod for sealing beads according to the present embodiment. (a)through (e) of FIG. 1 are views schematically illustrating a method forsealing beads according to the present invention, and show lateralcross-sectional views of an array 1.

The present embodiment deals with a case where beads 41 and 41′ aresealed into the array 1 including a lower layer section 10 and an upperlayer section 20. The lower layer section 10 includes a plurality ofreceptacles 13 each of which is capable of storing only one of the beads41 and 41′ and which are separated from each other by a side wall 12having a hydrophobic upper surface. Further, the upper layer section 20faces a surface of the lower layer section 10 on which surface thereceptacles 13 are provided.

Preferably, the beads have an average particle diameter of 1 μm to 4 μm.With this, the beads can be efficiently sealed into the array, and thearray can achieve high density. Note that the term “average particlediameter” herein refers to a value obtained as a result of measurementof the beads by means of electron microscope observation or dynamiclight scattering.

The present embodiment describes, but is not particularly limited to, acase of using beads specifically capturing target molecules. In thepresent embodiment, the beads to be sealed are a mixture of the beads41, which have not captured the target molecules yet, and the beads 41′,which have captured the target molecules.

For example, it is possible to use, as the beads specifically capturingthe target molecules, beads being bound to a molecule for specificallycapturing the target molecule. The molecule for specifically capturingthe target molecule may be bound to a modification group on a surface ofthe bead, e.g., via a linker. For example, the present invention may beconfigured such that the molecule for specifically capturing the targetmolecule is covalently bonded to an amino group on a surface of an aminogroup-modified bead via a crosslinker having N-hydroxysuccinimide and/orthe like.

The “target molecule” refers to a molecule which is to be detected(targeted molecule). Specifically, the “target molecule” herein refersto a molecule which is to be detected by causing the bead to capture themolecule. Examples of the target molecule encompass (i) biomoleculessuch as a protein, a nucleic acid, and sugar and (ii) virus particlesthemselves.

The molecule for specifically capturing the target molecule(hereinafter, such molecule is also referred to as a “target capturingmolecule”) may be chosen according to the target molecule. Examples ofthe target capturing molecule encompass a protein, an antibody, and anucleic acid. Preferably, one bead is bounded to hundred thousand ormore target capturing molecules. For example, in a case where the targetcapturing molecule is an antibody, the target capturing molecule has adissociation constant in nM order or so. However, with theabove-mentioned configuration, it is possible to cause the reactionbetween the beads and the target molecules with a sufficiently highconcentration of the target capturing (for example, in a case where theconcentration of the beads is 8×10⁶ particles/mL, the concentration ofthe target capturing molecules is approximately 1 nM).

The method for sealing beads according to the present embodimentincludes a step of beads introduction, a step of deaeration, and a stepof hydrophobic solvent introduction. Each of these steps will bedescribed in detail below.

(Step of Beads Introduction)

The following describes the step of beads introduction with reference to(a) and (b) of FIG. 1.

The step of beads introduction is a step of introducing a hydrophilicsolvent 42 containing the beads 41 and 41′ into a space 30 between thelower layer section 10 and the upper layer section 20. The hydrophilicsolvent 42 may be introduced into the space 30 between the lower layersection 10 and the upper layer section 20 along a direction which is inparallel with surfaces of the lower layer section 10 and the upper layersection 20, the surfaces of the lower layer section 10 and the upperlayer section 20 facing each other. For example, the hydrophilic solvent42 may be introduced into the space 30 via a through-hole (not shown)provided in at least one of the upper layer section 20 and the lowerlayer section 10.

Preferably used as the hydrophilic solvent 42 is, for example, at leastone selected from the group consisting of water, hydrophilic alcohol,hydrophilic ether, ketone, nitrile solvents, dimethyl sulfoxide (DMSO),and N,N-dimethylformamide (DMF) or is a mixture including the at leastone. Examples of hydrophilic alcohol encompass ethanol, methanol,propanol, and glycerin. Examples of hydrophilic ether encompasstetrahydrofuran, polyethylene oxide, and 1,4-dioxane. Examples of ketoneencompass acetone and methyl ethyl ketone. Examples of the nitrilesolvents encompass acetonitrile.

In addition to the beads 41 and 41′, the hydrophilic solvent 42 mayfurther include, e.g., a substance for specifically detecting the targetmolecule captured by any of the beads 41′. Such the substance may be,for example, a fluorescent substrate which liberates a fluorescentmaterial when decomposed by a certain enzyme bound to (i) the targetmolecule captured by any of the beads 41′ or (ii) a moleculespecifically bound to the target molecule. Examples of the moleculespecifically bound to the target molecule encompass a secondary antibodyand a nucleic acid. Examples of the certain enzyme encompassβ-galactosidase and peroxidase. Examples of the fluorescent substrateencompass fluorescein-di-β-galactopyranoside (FDG) and Amplex red(Registered Trademark).

(Step of Deaeration)

The following describes the step of deaeration with reference to (c) ofFIG. 1.

The step of deaeration is a step of deaerating the space 30 between thelower layer section 10 and the upper layer section 20, which is carriedout after the step of beads introduction and before the step ofhydrophobic solvent introduction. Preferably, the deaeration is carriedout by, for example, a method of allowing the array 1 to stand stillunder reduced pressure. Specifically, the deaeration is carried out by,for example, a method of allowing the array 1 to stand still in a vacuumdesiccator of approximately 0.1 atm for approximately 30 seconds.

The step of deaeration is not essential for the present invention.However, carrying out the step of deaeration removes the air in thereceptacles 13, thereby making it possible to efficiently introduce intothe receptacles 13 the hydrophilic solvent 42 containing the beads 41and 41′. This enables to efficiently seal the beads 41 and 41′ into thereceptacles 13. Therefore, it is preferable to carry out the step ofdeaeration.

(Step of Hydrophobic Solvent Introduction)

The following describes the step of hydrophobic solvent introductionwith reference to (d) and (e) of FIG. 1.

The step of hydrophobic solvent introduction is a step of introducing ahydrophobic solvent 43 into the space 30 between the lower layer section10 and the upper layer section 20. The step of hydrophobic solventintroduction is carried out after the step of beads introduction, andpreferably carried out after the step of deaeration.

The hydrophobic solvent 43 only needs to be a solvent that is difficultto be mixed with the hydrophilic solvent 42, which is used in the stepof beads introduction. Preferably used as the hydrophobic solvent 43 is,for example, at least one selected from the group consisting ofsaturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon,silicone oil, perfluorocarbon, halogen solvents, and hydrophobic ionicliquid or is a mixture including the at least one. Examples of saturatedhydrocarbon encompass alkane and cycloalkane. Examples of alkaneencompass decane and hexadecane. Examples of unsaturated hydrocarbonencompass squalene. Examples of aromatic hydrocarbon encompass benzeneand toluene. Examples of perfluorocarbon encompass Fluorinert(Registered Trademark) FC40 (available from SIGMA). Examples of thehalogen solvents encompass chloroform, methylene chloride, andchlorobenzene. The hydrophobic ionic liquid denotes ionic liquid whichis not dissociated at least in water. Examples of such the ionic liquidencompass 1-butyl-3-methylimidazolium hexafluorophosphate. The ionicliquid denotes a salt which is in the form of liquid at roomtemperature.

Carrying out the step of hydrophobic solvent introduction makes itpossible to efficiently form, in the respective receptacles 13, droplets(liquid droplets) covered with the hydrophobic solvent 43. Also,carrying out the step of hydrophobic solvent introduction makes itpossible to efficiently seal the beads 41 and 41′ into the droplets sothat any one of the beads 41 and 41′ is stored in each of the droplets.

According to the present embodiment, the beads 41 and 41′ are introducedthrough the space 30 between the lower layer section 10 and the upperlayer section 20, thereby enabling highly-efficient sealing of any oneof the beads into each of a large number of receptacles 13 which areprovided in a large area (e.g., an area of 1 cm² or more).

The present embodiment enables to provide a large-area droplet arrayincluding a large number of receptacles. For example, even with an arrayincluding one million or more receptacles, it is possible to efficientlyseal the beads 41 and 41′ into the receptacles so that any one of thebeads 41 and 41′ is stored in each of the receptacles. Thus, with thepresent embodiment, it is possible to detect the target molecules withhigh sensitivity, thereby enabling to detect the target molecules ofsuch a quite low concentration as approximately 0.1 aM.

[Method for Detecting Target Molecule]

Next, the following describes the method for detecting the targetmolecule according to the present embodiment.

The method for detecting the target molecule according to the presentembodiment includes a step of reaction, a step of sealing beads, and astep of determination.

The present embodiment uses, as the beads, beads that specificallycapture the target molecules. For example, each of such the beads may bethe one having been bound to a molecule for specifically capturing thetarget molecule. Suitably used as the beads, the target molecule, andthe molecule for specifically capturing the target molecule can be anyof those exemplified in the descriptions for the method for sealingbeads of the present embodiment.

The step of reaction is a step of reacting the beads with the targetmolecules. For example, the reaction between the beads and the targetmolecules can be carried out by mixing a solution containing the beadswith a solution containing the target molecules.

The step of sealing beads is a step of carrying out the above-mentionedmethod for sealing beads by use of the beads which have been reactedwith the target molecules in the step of reaction. Namely, the step ofsealing beads is (i) a step including the step of beads introduction andthe step of hydrophobic solvent introduction or (ii) a step includingthe step of beads introduction, the step of deaeration, and the step ofhydrophobic solvent introduction. Note that descriptions of the step ofbeads introduction, the step of deaeration, and the step of hydrophobicsolvent introduction are omitted here, since these steps can be carriedout in the same manner as those described in the above section “Methodfor bead sealing”.

The step of determination is a step of determining, after the step ofsealing beads, whether or not each of the receptacles 13 contains anyone of the beads 41′ having captured the target molecules.

Suitable examples of the method of determining whether or not each ofthe receptacles 13 contains any one of the beads 41′ having captured thetarget molecules encompass known molecular recognition reactions such asantigen-antibody reaction, streptavidin-biotin reaction, andcomplementary binding of nucleic acids. For example, this method can bea method of detecting a fluorescent material liberated from afluorescent substrate when decomposed by a certain enzyme bound to (i) atarget molecule or (ii) a molecule specifically bound to the targetmolecule. The detection of the fluorescent material is carried out by,for example, a method of determining a fluorescence intensity of eachreceptacle by use of, e.g., a fluorescence microscope or an imagesensor.

In the step of determination, it is preferable to also determine whethereach of the receptacles 13 contains any one of the beads 41 or any oneof the beads 41′. The determination of whether each of the receptacles13 contains any one of the beads 41 or any one of the beads 41′ can becarried out by, for example, microscopic observation to determine thepresence or absence of any one of the beads 41 or any one of the beads41′ in each of the receptacles 13. Alternatively, the determination ofthe presence or absence of any one of the beads 41 or any one of thebeads 41′ in each of the receptacles 13 can be carried out by a methodof detecting scattered light from the beads or a method of measuring anelectric potential with a field-effect transistor (FET).

After the step of determination, based on (i) the number of receptacles13 containing the beads 41 or the beads 41′ and (ii) the number ofreceptacles 13 containing the beads 41′ having captured the targetmolecules, it is possible to calculate a ratio of the number of beadshaving captured the target molecules with respect to the total number ofbeads. In this manner, it is possible to quantify a concentration of thetarget molecules.

According to the present embodiment, it is possible to provide alarge-area droplet array including a large number of receptacles;further, even with an array including one million or more receptacles,it is possible to efficiently seal the beads 41 and 41′ into thereceptacles. Thus, with the present embodiment, it is possible to detectthe target molecules with high sensitivity, thereby enabling to detectthe target molecules of such a quite low concentration as approximately0.1 aM.

[Array]

Next, the following describes a configuration of the array 1 of thepresent embodiment with reference to (a) of FIG. 1. The array 1 may bean array used in the method for sealing beads according to the presentembodiment, or may be an array used in the method for detecting thetarget molecule according to the present embodiment.

The array 1 includes the lower layer section 10 and the upper layersection 20.

The lower layer section 10 includes a plate-like member 11 and the sidewall 12 having a hydrophobic upper surface. The lower layer section 10includes the plurality of receptacles 13 that are separated from eachother by the side wall 12.

Preferably, the plate-like member 11 has a hydrophilic surface. The term“hydrophilic surface” refers to a surface whose affinity with ahydrophilic solvent is higher than that with a hydrophobic solvent. Theplate-like member 11 only needs to be made from a solid material. Forexample, the plate-like member 11 can be made from glass, silicon, or apolymer resin.

The side wall 12 is a structure that is provided on a surface of theplate-like member 11, preferably on the hydrophilic surface of theplate-like member 11, and is configured to separate the plurality ofreceptacles 13 from each other. The side wall 12 has the hydrophobicupper surface. The term “hydrophobic” herein is used as a synonym for“lipophilic”, and denotes a nature whose affinity with a hydrophobicsolvent is higher than that with a hydrophilic solvent.

Note that the side wall 12 needs to be configured such that its uppersurface, i.e., its surface facing the upper layer section 20, ishydrophobic. Whereas, a lateral surface of the side wall 12, i.e., aninner wall of each of the receptacles 13, may be either hydrophobic orhydrophilic.

For example, the side wall 12 may be made of a hydrophilic structure anda hydrophobic layer which is formed on an upper surface of thehydrophilic structure. The hydrophilic structure may be made from, e.g.,glass, silicon, or a polymer resin. The hydrophobic layer may be madefrom, e.g., a water repellent resin or a fluorocarbon polymer resin.Examples of the fluorocarbon polymer resin encompass amorphousfluorocarbon resin. The amorphous fluorocarbon resin is preferably used,because the amorphous fluorocarbon resin has a high hydrophobic propertyand has a low toxicity to a biological sample.

Preferable examples of the amorphous fluorocarbon resin encompass atleast one selected from CYTOP (Registered Trademark), TEFLON (RegisteredTrademark) AF2400, and TEFLON (Registered Trademark) AF1600. Amongthose, CYTOP (Registered Trademark) is most preferable, since it is easyto be microfabricated. CYTOP (Registered Trademark) has the followinggeneral formula:

TEFLON (Registered Trademark) AF2400 and TEFLON (Registered Trademark)AF1600 arepoly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene],which has the following general formula:

In AF2400, the dioxole component is 87 mol %, and in AF1600, it is 65mol %.

Alternatively, the side wall 12 may be made from a hydrophobic material.For example, the side wall 12 may be made from a fluorocarbon polymerresin or a paraxylene polymer resin. Examples of the fluorocarbonpolymer resin encompass an amorphous fluorocarbon resin. Preferably usedas the amorphous fluorocarbon resin is any of those exemplified above.

The side wall 12 only needs to have such a configuration that theplurality of receptacles 13 are provided on the plate-like member 11.For example, the side wall 12 may be a plate-like structure parts ofwhich corresponding to the receptacles 13 are holes.

A height (i.e., a thickness in a vertical direction) of the side wall 12measured from the surface of the plate-like member 11 only needs to bedesigned so that one of the beads 41 and 41′ contained in one of thereceptacles 13 would not be discharged therefrom during thelater-described step of hydrophobic solvent introduction. For example,the height of the side wall 12 may be designed so that most part of,preferably the whole part of, one of the beads 41 and 41′ contained inone of the receptacles 13 is positioned lower than the upper surface ofthe side wall 12.

In order to efficiently store the beads 41 and 41′ in the receptacles13, the height of the side wall 12 is preferably equal to or greaterthan the average particle diameter of the beads 41 and 41′. Further, inorder that only one of the beads 41 and 41′ is stored in one of thereceptacles 13, the height of the side wall 12 is preferably equal to orsmaller than 1.5 times the average particle diameter of the beads 41 and41′.

Each of the plurality of receptacles 13 is a recess capable of storingonly one of the beads 41 and 41′, and the plurality of receptacles 13are separated from each other by the side wall 12. Each of thereceptacles 13 has a bottom surface which is a part of the surface ofthe plate-like member 11, and the bottom surface is hydrophilic.

The receptacles 13 can have any shape or size, as long as the shape orsize allows each of the receptacles 13 to store only one of the beads 41and 41′ therein. A region surrounded by the bottom surface and thelateral surface of each of the receptacles 13 may be shaped in, e.g., acircular cylinder or a rectangular column.

A width “A” of each of the receptacles 13 in a horizontal direction(e.g., in a case where a cross section of each receptacle 13 when seenin the horizontal direction is shaped in a circle, the width “A” is adiameter of the circle; in a case where the cross section of eachreceptacle 13 when seen in the horizontal direction is shaped in asquare, the width “A” is a length of one side of the square) only needsto be larger than the average particle diameter of the beads 41 and 41′.Preferably, the width “A” is 1.5 to 2 times larger than the averageparticle diameter of the beads 41 and 41′, for example. In the presentembodiment, each of the receptacles 13 has a depth equal to the heightof the side wall 12. In order to efficiently store the beads in thereceptacles, the depth of each of the receptacles of the presentinvention is preferably equal to or greater than the average particlediameter of the beads. Further, in order that only one of the beads isstored in one of the receptacles, the depth of each of the receptaclesof the present invention is preferably equal to or smaller than 1.5times the average particle diameter of the beads.

According to the present embodiment, each of the receptacles 13 has thehydrophilic bottom surface, and the side wall 12 has the hydrophobicupper surface. This makes it possible to efficiently introduce thehydrophilic solvent 42 containing the beads 41 and 41′ into thereceptacles 13 in the later-described step of beads introduction, and toprevent the hydrophobic solvent 43 from entering the receptacles 13 inthe later-described step of hydrophobic solvent introduction. With this,the receptacles 13 storing the liquid droplets containing the beads 41and 41′ can be hermetically sealed with the hydrophobic solvent in anefficient manner.

The upper layer section 20 includes a plate-like member 21 and ahydrophobic layer 22. The hydrophobic layer 22 is provided on a surfaceof the plate-like member 21 which surface faces the lower layer section10. The plate-like member 21 is made from, e.g., glass, silicon, or apolymer resin. The hydrophobic layer 22 is made from, e.g., a waterrepellent resin or a fluorocarbon polymer resin. Examples of thefluorocarbon polymer resin encompass amorphous fluorocarbon resin.

The upper layer section 20 faces, via the space 30, the surface of thelower layer section 10 on which surface the receptacles 13 are provided.Namely, the space 30 exists between the side wall 12 and the hydrophobiclayer 22. The space 30 serves as a flow path. Thus, the array 1 isconfigured to have a flow cell structure.

The space 30 can be used as the flow path for allowing a fluid to flowbetween the lower layer section 10 and the upper layer section 20 in adirection in parallel with the surfaces of the lower layer section 10and the upper layer section 20, the surfaces of the lower layer section10 and the upper layer section 20 facing each other.

A distance between (i) the upper surface of the side wall 12 and (ii)the hydrophobic layer 22 the plate-like member 21, i.e., a width of thespace 30 in the vertical direction only needs to be larger than theaverage particle diameter of the beads 41 and 41′, and is preferably 10μm to 150 μm.

The lower layer section 10 or the upper layer section 20 may be providedwith the through-hole (not shown) through which the fluid is introducedinto the space 30. For example, the lower layer section 10 may have aregion provided with the receptacles 13 and a region provided with noreceptacles 13. Further, the lower layer section 10 may have thethrough-hole in the region provided with no receptacles 13;alternatively, the upper layer section 20 may have the through-hole in aregion facing the region of the lower layer section 10 provided with noreceptacles 13.

According to the present embodiment, an upper side of the space 30corresponds to the surface of the hydrophobic layer 22, and a lower sideof the space 30 corresponds to the upper surface of the side wall 12 andthe receptacles 13. Thus, except for parts of the space 30 correspondingto the bottom surfaces of the receptacles 13, the entire space 30 has ahydrophobic property. This configuration makes it possible toefficiently introduce the hydrophilic solvent 42 containing the beads 41and 41′ into the receptacles 13 in the later-described step of beadsintroduction. Further, this configuration prevents the hydrophobicsolvent 43 from entering the receptacles 13 in the later-described stepof hydrophobic solvent introduction. Thus, by introducing thehydrophobic solvent 43 into the space 30, it is possible to efficientlyform, in each of the receptacles 13, a droplet into which any one of thebeads 41 and 41′ is sealed.

The array 1 of the present embodiment may be, for example, an arrayincluding one million or more receptacles. Even with the array havingsuch a large area, the use of the method for sealing beads of thepresent embodiment or the method for detecting the target molecule ofthe present embodiment makes it possible to efficiently seal the beadsinto the receptacles so that any one of the beads is stored in each ofthe receptacles. Thus, according to the present embodiment, it ispossible to detect the target molecules with high sensitivity, therebyenabling to provide an array allowing detection of target molecules ofsuch a quite low concentration as approximately 0.1 aM.

[Kit]

Next, the following describes a configuration of a kit of the presentembodiment.

The kit of the present embodiment includes at least the array 1 and thebeads 41. Preferably used as the array 1 is the array 1 having theabove-described configuration. Each of the receptacles 13 in the array 1is configured to be capable of storing only one of the beads 41 includedin this kit.

Each of the beads 41 included in this kit may be the one specificallycapturing the target molecule. For example, each of the beads 41included in this kit may be the one having been bound to a molecule forspecifically binding to the target molecule. Suitably used as the targetmolecule and the molecule for specifically binding to the targetmolecule can be any of those mentioned above.

This kit may further include a substance for specifically detecting thetarget molecule. Preferably used as the substance for specificallydetecting the target molecule may be any of those mentioned above.Furthermore, the kit may further include, e.g., a water-soluble solventand/or a hydrophobic solvent.

[Target Molecule Detection Device]

Next, the following describes a target molecule detection device 50 ofthe present embodiment with reference to FIG. 2. FIG. 2 is a viewschematically illustrating one embodiment of a target molecule detectiondevice according to the present invention.

The target molecule detection device 50 of the present embodimentincludes the array 1, an image sensor 51, and a light source 52.Preferably used as the array 1 may be the one having the above-describedconfiguration, and therefore explanations of the array 1 are omittedhere.

The image sensor 51 is a sensor for detecting light emitted by each ofthe receptacles 13 when the beads having captured the target moleculesare stored in the receptacles 13. For example, the image sensor 51 maybe a sensor for detecting fluorescence emitted by a fluorescentsubstrate when decomposed by a certain enzyme bound to (i) the targetmolecule or (ii) a molecule specifically bound to the target molecule.Suitably used as the image sensor 51 can be, for example, a CMOS imagesensor.

The light source 52 is a light source for emitting light to the array 1.In FIG. 2, the light source 52 is provided above the array 1. However,the present invention is not particularly limited to this.Alternatively, the light source 52 may be the one emitting light to alateral side of the array 1, for example.

Between the array 1 and the image sensor 51, an interference filterand/or a light guide array may be provided, for example. Further,between the light source 52 and the array 1, an excitation filter may beprovided, for example.

According to the present embodiment, the array 1 and the image sensor 51are directly connected with each other. This makes it possible to easilydetermine, without use of other device such as a microscope, whether ornot any one of the beads having captured the target molecules is storedin each of the receptacles 13. This enables to carry out easy andhigh-speed detection of whether or not any one of the beads captured thetarget molecules is stored in each of the receptacles 13, and to providethe target molecule detection device at an affordable price.

The present application encompasses the following inventions.

A method for sealing beads of the present invention includes: (i) a stepof introducing a hydrophilic solvent containing beads into a spacebetween (a) a lower layer section including a plurality of receptacleseach of which is capable of storing only one of the beads and which areseparated from each other by a side wall having a hydrophobic uppersurface and (b) an upper layer section facing a surface of the lowerlayer section on which surface the plurality of receptacles areprovided; and (ii) a step of introducing a hydrophobic solvent into thespace, the step (ii) being carried out after the step (i).

Preferably, the method for sealing beads of the present inventionfurther includes (iii) a step of deaerating the space, the step (iii)being carried out after the step (i) and before the step (ii).

Preferably, according to the method for sealing beads of the presentinvention, the hydrophilic solvent is at least one selected from thegroup consisting of water, hydrophilic alcohol, hydrophilic ether,ketone, nitrile solvents, dimethyl sulfoxide, and N,N-dimethylformamide,or is a mixture including the at least one.

Preferably, according to the method for sealing beads of the presentinvention, the hydrophobic solvent is at least one selected from thegroup consisting of saturated hydrocarbon, unsaturated hydrocarbon,aromatic hydrocarbon, silicone oil, perfluorocarbon, halogen solvents,and hydrophobic ionic liquid, or is a mixture including the at leastone.

In order to attain the foregoing object, a method for detecting a targetmolecule of the present invention includes: (i) a step of reacting beadsspecifically capturing target molecules with the target molecules; (ii)a step of carrying out, by use of the beads, any of the above-mentionedmethods for sealing beads, the step (ii) being carried out after thestep (i); and (iii) a step of determining whether or not any one ofbeads having captured the target molecules is stored in each of theplurality of receptacles, the step (iii) being carried out after thestep (ii).

Preferably, according to the method for detecting a target molecule ofthe present invention, the beads are such beads to which moleculesspecifically bindable to the target molecules are bound.

An array of the present invention includes: a lower layer sectionprovided with a plurality of receptacles being separated from each otherby a side wall having a hydrophobic upper surface; and an upper layersection facing, via a space, a surface of the lower layer section onwhich surface the plurality of receptacles are provided.

Preferably, according to the array of the present invention, each of theplurality of receptacles has a hydrophilic bottom surface.

Preferably, according to the array of the present invention, the upperlayer section has a hydrophobic surface facing the lower layer section.

Preferably, according to the array of the present invention, at leastone of the upper layer section and the lower layer section has athrough-hole via which a fluid is introduced into the space.

In order to attain the foregoing object, a kit of the present inventionincludes: any of the above-mentioned arrays; and beads, each of theplurality of receptacles being capable of storing only one of the beads.

In order to attain the foregoing object, a target molecule detectiondevice of the present invention includes: any one of the above-mentionedarrays; and an image sensor for detecting light being emitted from eachof the plurality of receptacles in a case where beads having capturedtarget molecules are stored in the plurality of receptacles.

The present invention is not limited to the description of theembodiments above, but may be altered by a skilled person within thescope of the claims. The embodiments of the present invention aredescribed in further detail via the following Examples. Needless to say,the present invention is not limited to these Examples. The inventionbeing thus described, it will be clear that the same may be varied inmany ways.

EXAMPLES

The following will describe materials and methods that were used inExamples.

(Materials)

In the Examples, used as the target molecule was streptavidin (purchasedfrom SIGMA) labeled with β-galactosidase (hereinafter, also simplyreferred to as “streptavidin”). Further, used as the beads werebiotinylated beads prepared by biotinylating amino beads having anaverage particle diameter of 3 μm (material: polystyrene; micromer-NH₂-3μm; purchased from Micromod).

(Preparation of Biotinylated Beads)

By the following method, amino groups of amino beads were reacted withNHS-PEO4-Biotin, so that the amino beads were biotinylated.

First, 750 μL of buffer A (100 mM phosphoric acid buffer, pH 8.0) wasadded to 250 μL of amino beads.

Next, the resultant was subjected to centrifugation at 10000 rpm at 4°C. for 10 minutes, so that the amino beads were gathered up andcollected. Then, the amino beads were suspended in 500 μL of buffer A(suspension A). Thereafter, 50 μL of NHS-PEO4-Biotin (2 μg/50 μL DMSO)was added to the suspension A, and then the amino beads andNHS-PEO4-Biotin were reacted under gentle stirring at 25° C. for atleast 3 hours (the tube was rotated end-over-end for mixing).

Next, biotinylated beads thus obtained were washed. The mixture wassubjected to centrifugation at 10000 rpm at 4° C. for 10 minutes, sothat the biotinylated beads were gathered up and collected. Then, anaqueous phase therein was removed by a Pipetman pipette. To theresulting precipitate of the biotinylated beads, 1 mL of buffer A wasadded so that the precipitate of the biotinylated beads was suspended.This process was repeated six times, so that unreacted NHS-PEO4-Biotinwas removed. Then, the resultant was suspended in 500 μL of buffer A(suspension B). The suspension B was preserved at 4° C.

Next, a concentration of the biotinylated beads in the suspension B wasmeasured. The number of biotinylated beads in a certain volume wascounted by use of a hemacytometer, so that the concentration of thebiotinylated beads was found (approximately 3.0×10⁸ beads/mL). In orderthat the number of biotinylated beads was easily counted, the countingwas carried out after the suspension B was diluted by the buffer A byapproximately 5-fold, for example.

By the above-mentioned method, the biotinylated beads were obtained.

(Capturing of Streptavidin)

Next, by the following method, streptavidin was captured by use of thebiotinylated beads.

First, the biotinylated beads were diluted (8×10⁶ beads/500 μL).Further, streptavidin labeled with β-galactosidase was diluted by thebuffer B (100 mM phosphoric acid buffer, pH 8.0, containing 0.1% TWEEN20(detergent)) so that a concentration of streptavidin became two timeshigher than a target concentration (total amount: 500 μL).

Then, 500 μL of the biotinylated beads and 500 μL of streptavidin thusdiluted were mixed together in a tube (total amount: 1 mL). The tube wasshaken vertically in a gentle manner, so that reaction between thebiotinylated beads and streptavidin was carried out at 25° C. for 30minutes.

Next, the resultant was subjected to centrifugation at 10000 rpm at 4°C. for 10 minutes, so that the beads after the reaction (a mixture of(i) complexes of streptavidin and the biotinylated beads and (ii)unreacted biotinylated beads) were gathered up and collected. Then, anaqueous phase therein was removed by a Pipetman pipette. To theresulting precipitate of beads, 1 mL of buffer A was added andsuspended. This process was repeated four times for washing, so thatunreacted target molecules were removed.

Next, to the precipitate of the beads after the washing, 15 μL of bufferC (100 mM phosphoric acid buffer, pH 7.5, 1 mM MgCl₂) was added forsuspension. An ultimate concentration of the beads was approximately6.5×10⁶ beads/15-μL buffer C.

(Production of Array)

Next, by the following method, an array having the same flow cellstructure as that of the array 1 shown in (a) through (e) of FIG. 1 wasproduced. In the following descriptions, members having the samefunctions as those in the array 1 are given the same reference signs.

First, a hydrophilic-hydrophobic patterned glass (lower layer section10) and an upper side glass (upper layer section 20) (height: 24mm×width: 26 mm×depth: 5 mm, SiO₂, with a through-hole having a diameterof 1 mm) were prepared.

(Preparation of Hydrophilic-Hydrophobic Patterned Glass)

With reference to FIG. 7, the following describes a specific method forpreparing the hydrophilic-hydrophobic patterned glass. FIG. 7 is a viewfor explaining a method for preparing a hydrophilic-hydrophobicpatterned glass according to an example of the present invention.

According to the present embodiment, photolithography and dry etchingwere carried out so that a hydrophilic-hydrophobic pattern was formed onglass. In order to form the hydrophilic-hydrophobic pattern, three stepsincluding a step of CYTOP (Registered Trademark) application, a step ofphotolithography, and a step of etching and resist removal were carriedout.

In the step of CYTOP (Registered Trademark) application, CYTOP(Registered Trademark) CTL-809 (product name; available from ASAHIGLASS) was first applied onto glass of 24 mm (height)×32 mm (width)(product name: NEO MICRO COVER GLASS Thickness No. 1; available fromMATSUNAMI) (plate-like member 11), so that a hydrophobic resin layer 61was formed.

Next, in the step of photolithography, a positive photoresist 62(product name: AZ-4903; available from AZ Electronic Materials USA) wasapplied onto the hydrophobic resin layer 61. Next, via a photomask 63having a desired pattern, the resultant was exposed to UV emitted fromabove, so that an alkaline development process was carried out. As aresult of the development process, the photoresist 62 was dissolved onlyin parts irradiated with UV, so that parts of the hydrophobic resinlayer 61 which parts faced the parts of the photoresist 62 irradiatedwith UV were exposed.

After that, in the step of etching and resist removal, the glass wasetched by O₂ plasma via a partially dissolved photoresist 62′, so thatthe parts of the resin layer 61 were removed. As a result, a hydrophobicside wall 12 was obtained. Finally, the photoresist 62′ was dissolved byan organic solvent. Thus, the hydrophilic-hydrophobic pattern wasobtained.

Further detailed procedures for the above process are described below.The reference numerals (1) through (23) in FIG. 7 correspond to (1)through (23) below, respectively.

<Step of CYTOP (Registered Trademark) Application (i.e., Preparation ofCYTOP (Registered Trademark) Layer Having Film Thickness ofApproximately 3.3 μm to 3.5 μm by the Following Procedures)>

(1) First, glass (plate-like member 11) was washed and CYTOP (RegisteredTrademark) CTL-809 was applied onto the glass.

(2) Next, the glass was immersed in 10N KOH overnight.

(3) The cover glass having been immersed in KOH was washed withdeionized water ten or more times.

(4) The glass was dried with a hot plate at 180° C.

(5) The glass thus dried was cooled to room temperature.

(6) Approximately 70 μL of CYTOP (Registered Trademark) CTL-809 waspoured onto the glass.

(7) Spin-coating was carried out according to the following program A:

[Program A]

Slope: 5 seconds

500 rpm: 10 seconds

Slope: 5 seconds

2000 rpm: 30 seconds

Slope: 5 seconds

End

(8) The glass was baked on the hot plate at 180° C. for an hour.

By repeating the above procedures (6) through (8) four times, ahydrophobic resin layer 61 having a depth of 3.3 μm to 3.5 μm wasobtained.

<Step of Photolithography>

Next, photolithography was carried out.

(9) Onto the resin layer 61 prepared by the step of CYTOP (RegisteredTrademark) application, a positive photoresist 62 (AZ-4903) was pouredin such an amount that the poured positive photoresist 62 spread on theglass so as to be in a diameter of approximately 8 mm.

(10) Spin-coating was carried out according to the following program B:

[Program B]

Slope: 5 seconds

500 rpm: 10 seconds

Slope: 5 seconds

4000 rpm: 60 seconds

Slope: 5 seconds

End

(11) The photoresist remaining on an edge of the glass was wiped outwith a piece of gauze dampened with 100% EtOH.

(12) The glass was baked at 55° C. for 3 minutes.

(13) The glass was baked at 110° C. for 5 minutes.

(14) A photomask 63 was washed with acetone, and then the photomask 63was set in a mask aligner (available from SAN-EI ELECTORIC).

(15) The glass to which the photoresist 62 was applied was set on asample table of the mask aligner, and the sample table was lifted up, sothat the glass and the photomask 63 were brought into contact with eachother.

(16) The glass thus brought into contact with the photomask 63 wasirradiated with UV for 35 seconds (power: 256).

(17) The glass was immersed in AZ Developer (available from AZElectronic Materials USA) for 5 minutes or more for development.

(18) The glass was rinsed with MilliQ (distilled water) forapproximately 10 minutes.

<Step of Etching and Resist Removal>

Subsequently, etching and removal of the resist were carried out.

(19) The glass was subjected to O₂ plasma etching by use of RIE-10NR(available from Samco) under certain process conditions (O₂: 50 sccm,pressure: 10 Pa, power: 50 W, time: 30 min.).

(20) The glass having been subjected to the etching was immersed inacetone, and then the glass was sonicated for 15 minutes.

(21) Acetone was exchanged for new one, and then the glass was sonicatedagain for 15 minutes.

(22) The glass was sonicated in EtOH for 15 minutes.

(23) The glass was washed with MilliQ (distilled water).

In the above-described method, a plurality of wells (receptacles 13)were formed on the glass. A region surrounded by the bottom surface andthe lateral surface of each of the wells was shaped in a circularcylinder. A cross section of each well in the horizontal direction wasshaped in a circle having a diameter of 5 μm. A height of the side wall,by which the wells were partitioned, was approximately 3.3 μm to 3.5 μm.Further, a distance “B”, by which two adjacent wells were separated fromeach other, was 5 μm.

(Preparation of Upper Side Glass)

In the following method, an upper side glass was prepared. In order toprepare the upper side glass, such glass was used that has a thicknessof 5 mm and a through-hole having a diameter of 1 mm. One side of thisglass was covered with approximately 70 μL of CYTOP (RegisteredTrademark) CTL-809 (product name; available from ASAHI GLASS). Then,spin-coating was carried out according to the following program C:

[Program C]

Slope: 5 seconds

500 rpm: 10 seconds

Slope: 5 seconds

2000 rpm: 30 seconds

Slope: 5 seconds

End

Thereafter, the glass was baked on a hot plate at 180° C. for an hour.

In the above-described method, an upper side glass having one sideprovided with a hydrophobic layer of a thickness of 1 μm was prepared.

(Bonding of Hydrophilic-Hydrophobic Patterned Glass and Upper SideGlass)

Next, high vacuum grease (available from DOW CORNING TORAY) was appliedto a piece of backing paper of Parafilm (available from Peckiney PlasticPackaging), and then the piece of backing paper of Parafilm was attachedonto a part of the hydrophilic-hydrophobic patterned glass, the partbeing on a side on which the hydrophilic-hydrophobic pattern was formed,and the part not having the hydrophilic-hydrophobic pattern. The upperside glass was bonded to the side of the hydrophilic-hydrophobicpatterned glass on which side the hydrophilic-hydrophobic pattern wasformed, in such a manner that the coating agent-coated side of the upperside glass faced the hydrophilic-hydrophobic patterned glass.

Consequently, a space was made between the hydrophilic-hydrophobicpatterned glass and the upper side glass. A width of this space in thevertical direction, i.e., a distance between (i) the upper surface ofthe side wall of the hydrophilic-hydrophobic patterned glass and (ii)the upper side glass was approximately 150 μm.

(Sealing of Beads into Droplets)

Next, in the following method, the beads having been reacted withstreptavidin were sealed into droplets.

First, 50 mM fluorescein-di-β-galactopyranoside (FDG) (available fromMarker Gene Technology)/DMSO was diluted with FDG buffer (100 mM KPibuffer (PH=7.5), 1 mM MgCl₂, 4 μL/mL 2-mercaptethanol), so that 4 mM FDGsolution was prepared. Then, 15 μL of the beads (6.5×10⁶ beads/15-μLbuffer C) and 15 μL of 4 mM FDG solution were mixed together, so that abeads solution was prepared.

Next, 30 μL of the beads solution was loaded into the flow path by ayellow tip via the through-hole of the upper side glass (see (a) and (b)of FIG. 1).

Next, in order to remove the air in the wells, deaeration was carriedout for one minute (see (c) of FIG. 1). The deaeration was carried outin such a manner that the array was allowed to stand still in a vacuumdesiccator of approximately 0.1 atm for approximately 30 seconds. Afterthat, the array was left at rest for approximately 5 minutes, so thatthe beads were precipitated into the bottoms of the wells.

Thereafter, 200 μL to 1000 μL of Fluorinert (Registered Trademark) FC40(available from SIGMA) was loaded into the flow path via thethrough-hole of the upper side glass (see (d) and (e) of FIG. 1).

As a result, an aqueous phase was trapped only in the wells, so thatdroplets were formed. Thus, the beads were sealed into the droplets.

Example 1

By the above-described method, biotinylated beads and 1 fM streptavidinwere reacted with each other, and then the resultant was introduced intoan array together with FDG, so that the beads were sealed into droplets,respectively. The array used in Example 1 was a 1.0 cm×1.0 cm arrayincluding a total of 1097600 receptacles, specifically, including a20×20 (horizontally and vertically) matrix of subarrays each (i) havinga size of 512 μm×512 μm and (ii) including 2744 receptacles. This arraywas observed with a fluorescence microscope (IX71 (available fromOLYMPUS)).

FIG. 3 shows a fluorescence image of the array into which the beads weresealed in one example of the present invention. What is shown in FIG. 3is one subarray. As shown in FIG. 3, some bright points were observed inthe fluorescence image (138 bright points in one field). These brightpoints indicate positions of receptacles into which biotinylated beadshaving captured streptavidin were sealed. This shows that the use of themethod of the present invention makes it possible to adequately detect 1fM streptavidin.

Comparative Example 1

In a comparative example of the present invention, target molecules weredetected by a conventional bulk measurement method.

12.5 fM streptavidin, which is 12.5 times higher concentration than that(1 fM) in Example 1, was mixed with FDG, and the resultant was measuredfor fluorescence by a fluorescence spectrophotometer. Further, a controlexperiment was carried out in the same manner by use of 6.3 pMstreptavidin, which is 500 times higher concentration than that of thiscomparative example.

FIG. 4 shows results of the comparative example and the controlexperiment. FIG. 4 is a graph showing fluorescence intensities observedwhen the target molecules were detected by the conventional method. InFIG. 4, a graph line “a” shows a result of the case involving use of12.5 fM streptavidin, whereas a graph line “b” shows a result of thecase involving use of 6.3 pM streptavidin.

As shown in FIG. 4, in the case where the conventional method was used,it was impossible to detect 12.5 fM streptavidin. This shows that 12.5fM is lower than a detection limit of the conventional method.

Example 2

Next, biotinylated beads were reacted with streptavidin of fivedifferent concentrations (1 fM, 100 aM, 10 aM, 1 aM, and 0.1 aM), andthen were introduced into arrays together with FDG, so that the beadswere sealed into droplets, respectively. Each of the arrays used inExample 2 had the same configuration as that used in Example 1. Each ofthese arrays was observed in a bright field image and in a fluorescenceimage by a microscope.

(Detection Results)

(a) through (f) of FIG. 5 show microscopic images of the arrays intowhich the beads were sealed in another example of the present invention.Note that each of (a), (c), and (e) of FIG. 5 shows a bright field imageof a respective one of subarrays, whereas each of (b), (d), and (f) ofFIG. 5 shows a fluorescence image of a respective one of the subarraysshown in (a), (c), and (e) of FIG. 5. Further, (a) and (b) of FIG. 5show the results obtained in the case involving the use of 1 fMstreptavidin; (c) and (d) of FIG. 5 show the results obtained in thecase involving the use of 100 aM streptavidin; and (e) and (f) of FIG. 5show the results obtained in the case involving the use of 10 aMstreptavidin.

In the case involving the use of 1 fM streptavidin, a total number ofbeads sealed into one subarray was 1735; among those beads, the numberof beads having captured streptavidin was 138. In the case involving theuse of 100 aM streptavidin, a total number of beads sealed into onesubarray was 2008; among those beads, the number of beads havingcaptured streptavidin was 6. In the case involving the use of 10 aMstreptavidin, a total number of beads sealed into one subarray was 1360;among those beads, the number of beads having captured streptavidin was1.

(Comparison Between Theoretical Value and Experimental Value)

Further, a theoretical value and an experimental value of a ratio (%) ofthe number of beads (active beads) having captured streptavidin withrespect to the total number of beads were calculated for each of thestreptavidin concentrations.

Calculated as the theoretical value was a ratio (%) of the number ofstreptavidin molecules with respect to the total number of beads used inthe reaction with streptavidin. Whereas, calculated as the experimentalvalue was a ratio (%) of the number of beads having capturedstreptavidin with respect to the number of beads stored in the array.

FIG. 6 shows a graph illustrating a relationship, observed in saidanother example of the present invention, between (i) a concentration ofstreptavidin and (ii) a ratio of the number of beads having capturedstreptavidin with respect to the number of beads stored in the array. InFIG. 6, a graph line “a” shows the theoretical values, a circular dotshows the experimental value, and a circle drawn with a dotted lineshows averages of the experimental values (N=2 to 3). Further, a graphline “b” is a line by which averages of the experimental values wereapproximated.

As shown in FIG. 6, the theoretical values and the experimental valuesare almost the same as each other. This shows that the method of thepresent example has a high quantitative accuracy, and is capable ofaccurately measuring a concentration of target molecules. These resultsshow that the method of the present example makes it possible toadequately detect even target molecules of 0.1 aM or so.

Example 3

By the above-described method, a beads solution (6.5×10⁶ beads/30 μL)was introduced (loaded) into the array having the flow cell structureprepared in Example 1, so that the beads were sealed (trapped) intodroplets. Then, a trapping efficiency (i.e., a ratio (%) of the numberof trapped beads with respect to the number of loaded beads) during thisprocess was calculated.

A result of the calculation is shown in FIG. 8. FIG. 8 is a viewillustrating (i) a bead trapping efficiency found in a case involvingthe use of the array having the flow cell structure (Example 3) and (ii)a bead trapping efficiency found in a case involving the use of an arraynot having the flow cell structure (Comparative Example 2).

Comparative Example 2

In this comparative example, the array not having the flow cellstructure (i.e., the array made of the above-describedhydrophilic-hydrophobic patterned glass only) was used for beads sealing(amino beads of Φ=3 μm; micromer-NH₂-3 μm; available from micromod).

By the below-described method, beads were sealed into thehydrophilic-hydrophobic patterned glass by use of a beads solution whichwas diluted at the same concentration (2.2×10⁸ beads/mL=6.5×10⁶ beads/30μL) as that used in the case involving the use of the array having theflow cell structure (Example 3).

(1) The beads were diluted at a concentration of 2.2×10⁸ beads/mL with abuffer (100 mM KPi buffer (PH=7.5), 1 mM MgCl₂, 2 μL/mL2-mercaptethanol).

(2) The hydrophilic-hydrophobic patterned glass (prepared by theabove-mentioned method) was bonded to a bottom of a petri dish (35-mmpetri dish, available from Becton Dickinson). An adhesive used thereforwas Araldite AR-R30 (available from NICHIBAN).

(3) An upper surface of the hydrophilic-hydrophobic patterned glass wascovered with 500 μL of the beads solution.

(4) The resultant was subjected to deaeration, and then was incubatedfor 5 minutes.

(5) 2 mL of FC40 (Fluorinert (Registered Trademark) FC40, available fromSIGMA) was loaded onto the hydrophilic-hydrophobic patterned glass, sothat the beads were sealed thereinto.

(6) In order to prevent evaporation of the droplets, water wasintroduced thereonto so that an oil phase was covered with the water.

After that, the number of beads confined in the droplets was counted,and then a trapping efficiency (a ratio (%) of the number of trappedbeads with respect to the number of loaded beads) was calculated.

Results of the calculations are shown in FIG. 8. As shown in FIG. 8, thetrapping efficiency found in the case involving the use of the arrayhaving the flow cell structure was 25 or more times higher than that inthe case involving the use of the array not having the flow cellstructure. The reason for this is considered as follows: In the caseinvolving the use of the array not having the flow cell structure, adistance in which the beads could scatter in a vertical direction wasincreased, which made it difficult for the beads to come closer to thesubstrate.

Further, in the case involving the use of the array not having the flowcell structure, the whole surface of the substrate of the array needs tobe covered with the beads solution. This requires a large amount ofbeads solution (i.e., the beads solution whose amount is approximately16 times larger than that used in the case involving the use of thearray having the flow cell structure). Thus, the use of the array havingthe flow cell structure makes it possible to carry out sealing of beadswith a small amount of beads solution.

INDUSTRIAL APPLICABILITY

The present invention is suitably applicable to a method for detectingtarget molecules of low concentration, an array therefor, a devicetherefor, and the like.

REFERENCE SIGNS LIST

-   -   1 Array    -   10 Lower layer section    -   20 Upper layer section    -   12 Side wall    -   13 Receptacle    -   30 Space    -   41, 41′ Beads    -   42 Hydrophilic solvent    -   43 Hydrophobic solvent

The invention claimed is:
 1. A detecting method using a kit and adeaerating device, wherein said kit comprises: an array having a flowcell structure, said array including: a lower layer section providedwith a plurality of receptacles being separated from each other by aside wall having a hydrophobic upper surface; and an upper layer sectionfacing, via a space, a surface of the lower layer section on whichsurface the plurality of receptacles are provided; beads; a hydrophilicsolvent; and a hydrophobic solvent; each of the plurality of receptaclesis capable of storing Only one of the beads; and said space is used as aflow path for allowing a fluid to flow between the lower layer sectionand the upper layer section; and said deaerating device is set forremoving air in the plurality of receptacles while keeping thehydrophilic solvent as it is in the plurality of receptacles; said kitand said deaerating device being used to carry out a method for sealingthe beads, the method comprising the steps of: (i) introducing thehydrophilic solvent containing the beads into the space between thelower layer section and the upper layer section; (ii) deaerating toremove air in the plurality of receptacles while keeping the hydrophilicsolvent as it is in the plurality of receptacles; and (iii) introducingthe hydrophobic solvent into the space to flow and to displace thehydrophilic solvent; wherein step (ii) is carried out between step (i)and step (iii).
 2. The detecting method according to claim 1, whereineach of the plurality of receptacles has a hydrophilic bottom surface.3. The detecting method according to claim 2, wherein the upper layersection has a hydrophobic surface facing the lower layer section.
 4. Thedetecting method according to claim 3, wherein at least one of the upperlayer section and the lower layer section has a through-hole via which afluid is introduced into the space.
 5. The detecting method according toclaim 2, wherein at least one of the upper layer section and the lowerlayer section has a through-hole via which a fluid is introduced intothe space.
 6. The detecting method according to claim 1, wherein theupper layer section has a hydrophobic surface facing the lower layersection.
 7. The detecting method according to claim 6, wherein at leastone of the upper layer section and the lower layer section has athrough-hole via which a fluid is introduced into the space.
 8. Thedetecting method according to claim 1, wherein at least one of the upperlayer section and the lower layer section has a through-hole via which afluid is introduced into the space.
 9. The detecting method according toclaim 1, wherein each of the plurality of receptacles is made byphotolithography and dry etching.
 10. The detecting method according toclaim 1, wherein a nucleic acid is captured by the bead sealed in theplurality of receptacles.
 11. The detecting method according to claim 1,wherein said side wall is made of an amorphous fluorocarbon resin. 12.The detecting method according to claim 1, wherein a width of said spacein the vertical direction is from 10 μm to 150 μm.